A microRNA396b-growth regulating factor module controls castor seed size by mediating auxin synthesis.

Castor (Ricinus communis L.) is an importance crop cultivated for its oil and economic value. Seed size is a crucial factor that determines crop yield. Gaining insight into the molecular regulatory processes of seed development is essential for the genetic enhancement and molecular breeding of castor. Here, we successfully fine-mapped a major QTL related to seed size, qSS3, to a 180 kb interval on chromosome 03 using F2 populations (DL01×WH11). A 17.6-kb structural variation (SV) was detected through genomic comparison between DL01 and WH11. Analysis of haplotypes showed that the existence of the complete 17.6 kb structural variant may lead to the small seed characteristic in castor. In addition, we found that qSS3 contains the microRNA396b (miR396b) sequence, which is situated within the 17.6 kb SV. The results of our experiment offer additional evidence that miR396-Growth Regulating Factor 4 (GRF4) controls seed size by impacting the growth and multiplication of seed coat and endosperm cells. Furthermore, we found that RcGRF4 activates the expression of YUCCA6 (YUC6), facilitating the production of IAA in seeds and thereby impacting the growth of castor seeds. Our research has discovered a crucial functional module that controls seed size, offering a fresh understanding of the mechanism underlying seed size regulation in castor.

Regulation of capsule spine formation in castor.

Castor (Ricinus communis L.) is a dicotyledonous oilseed crop that can have either spineless or spiny capsules. Spines are protuberant structures that differ from thorns or prickles. The developmental regulatory mechanisms governing spine formation in castor or other plants have remained largely unknown. Herein, using map-based cloning in 2 independent F2 populations, F2-LYY5/DL01 and F2-LYY9/DL01, we identified the RcMYB106 (myb domain protein 106) transcription factor as a key regulator of capsule spine development in castor. Haplotype analyses demonstrated that either a 4,353-bp deletion in the promoter or a single nucleotide polymorphism leading to a premature stop codon in the RcMYB106 gene could cause the spineless capsule phenotype in castor. Results of our experiments indicated that RcMYB106 might target the downstream gene RcWIN1 (WAX INDUCER1), which encodes an ethylene response factor known to be involved in trichome formation in Arabidopsis (Arabidopsis thaliana) to control capsule spine development in castor. This hypothesis, however, remains to be further tested. Nevertheless, our study reveals a potential molecular regulatory mechanism underlying the spine capsule trait in a nonmodel plant species.

The double flower variant of yellowhorn is due to a LINE1 transposon-mediated insertion.

As essential organs of reproduction in angiosperms, flowers, and the genetic mechanisms of their development have been well characterized in many plant species but not in the woody tree yellowhorn (Xanthoceras sorbifolium). Here, we focused on the double flower phenotype in yellowhorn, which has high ornamental value. We found a candidate C-class gene, AGAMOUS1 (XsAG1), through bovine serum albumin sequencing and genetics analysis with a Long Interpersed Nuclear Elements 1 (LINE1) transposable element fragment (Xsag1-LINE1-1) inserted into its second intron that caused a loss-of-C-function and therefore the double flower phenotype. In situ hybridization of XsAG1 and analysis of the expression levels of other ABC genes were used to identify differences between single- and double-flower development processes. These findings enrich our understanding of double flower formation in yellowhorn and provide evidence that transposon insertions into genes can reshape plant traits in forest trees.

Theoretical investigation of the reaction mechanism of THP oxidative rearrangement catalysed by BBOX.

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme FeII/2OG dependent enzyme that is able to perform two different kinds of catalytic reactions on 3-(2,2,2-trimethylhydrazinium) propionate (THP) to produce distinct catalytic products. Although the structure of BBOX complexed with THP has been resolved, the details of its catalytic mechanism are still elusive. In this study, by employing molecular dynamics (MD) simulations and density functional theory (DFT) calculations, the mechanism of the THP oxidative rearrangement reactions catalysed by BBOX was investigated. Our calculations revealed how the enzyme undergoes a conformational conversion to initiate the catalytic reactions. In the first catalytic step, BBOX performs hydrogen abstraction from the substrate THP as a common non-heme iron enzyme. Due to the structure of the substrate stabilizing the radical species and polarizing the adjacent N-N bond, in the next step, THP takes the pathway for N-N bond homolysis but not regular hydroxyl rebounding. The cleaved ammonium radical could either react with the hydroxyl group on the iron centre of the enzyme or recombine with the other cleaved fragment of the substrate to generate the rearranged product. This study revealed the catalytic mechanism of BBOX, detailing how the enzyme and the substrate regulated the hydroxyl rebound process to generate various products.

Association of Molar Incisor Hypomineralization with Hypomineralized Second Primary Molars: An Updated Systematic Review with a Meta-Analysis and Trial Sequential Analysis.

INTRODUCTION: There is a correlation between molar incisor hypomineralization (MIH) and hypomineralized second primary molars (HSPM), but this relationship has not been definitively confirmed. The purpose of this systematic review and meta-analysis was to reevaluate whether children with HSPM are more affected by MIH than non-HSPM children. METHODS: A systematic search was conducted in four databases (PubMed, Embase, Web of Science, and the Cochrane Library) for literature, published up to December 2022. Two independent reviewers conducted the study search and screening, quality assessment, and data extraction according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The risk-of-bias assessment of all included cohort studies and case-control studies was assessed by the Newcastle-Ottawa Scale (NOS), and cross-sectional studies were assessed using the Agency for Healthcare Research Quality (AHRQ) scale. RevMan 5.4 software was used for all data analyses, with odds ratios (ORs) and 95% confidence intervals (CIs) as the effect measures. Sensitivity and subgroup analyses were conducted to identify the potential sources of heterogeneity among the studies. Publication bias was tested and corrected by funnel plots and Egger's test. Trial sequential analysis (TSA) was performed using TSA 0.9.5.10 Beta software to control for type-1 and type-2 errors. RESULTS: A total of 12 studies involving 8,944 children were included in this meta-analysis. Compared with the non-HSPM group, the HSPM group had an increased likelihood of MIH (OR = 10.90, 95% CI = 4.59-25.89, p < 0.05). All the included studies were of moderate-to-high quality. TSA and sensitivity analyses suggested the robustness of this outcome. CONCLUSION: This systematic review demonstrated a certain correlation between HSPM and MIH, suggesting that HSPM can play a predictive role in the occurrence of MIH. Further high-quality, multicenter, and large-sample longitudinal studies are highly recommended.

The microglial innate immune receptors TREM-1 and TREM-2 in the anterior cingulate cortex (ACC) drive visceral hypersensitivity and depressive-like behaviors following DSS-induced colitis.

Inflammatory bowel disease (IBD) is a chronic condition with a high recurrence rate. To date, the clinical treatment of IBD mainly focuses on inflammation and gastrointestinal symptoms while ignoring the accompanying visceral pain, anxiety, depression, and other emotional symptoms. Evidence is accumulating that bi-directional communication between the gut and the brain is indispensable in the pathophysiology of IBD and its comorbidities. Increasing efforts have been focused on elucidating the central immune mechanisms in visceral hypersensitivity and depression following colitis. The triggering receptors expressed on myeloid cells-1/2 (TREM-1/2) are newly identified receptors that can be expressed on microglia. In particular, TREM-1 acts as an immune and inflammatory response amplifier, while TREM-2 may function as a molecule with a putative antagonist role to TREM-1. In the present study, using the dextran sulfate sodium (DSS)-induced colitis model, we found that peripheral inflammation induced microglial and glutamatergic neuronal activation in the anterior cingulate cortex (ACC). Microglial ablation mitigated visceral hypersensitivity in the inflammation phase rather than in the remission phase, subsequently preventing the emergence of depressive-like behaviors in the remission phase. Moreover, a further mechanistic study revealed that overexpression of TREM-1 and TREM-2 remarkably aggravated DSS-induced neuropathology. The improved outcome was achieved by modifying the balance of TREM-1 and TREM-2 via genetic and pharmacological means. Specifically, a deficiency of TREM-1 attenuated visceral hyperpathia in the inflammatory phase, and a TREM-2 deficiency improved depression-like symptoms in the remission phase. Taken together, our findings provide insights into mechanism-based therapy for inflammatory disorders and establish that microglial innate immune receptors TREM-1 and TREM-2 may represent a therapeutic target for the treatment of pain and psychological comorbidities associated with chronic inflammatory diseases by modulating neuroinflammatory responses.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

The Characterization of the Phloem Protein 2 Gene Family Associated with Resistance to Sclerotinia sclerotiorum in Brassica napus.

In plants, phloem is not only a vital structure that is used for nutrient transportation, but it is also the location of a response that defends against various stresses, named phloem-based defense (PBD). Phloem proteins (PP2s) are among the predominant proteins in phloem, indicating their potential functional role in PBD. Sclerotinia disease (SD), which is caused by the necrotrophic fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum), is a devastating disease that affects oil crops, especially Brassica napus (B. napus), mainly by blocking nutrition and water transportation through xylem and phloem. Presently, the role of PP2s in SD resistance is still largely estimated. Therefore, in this study, we identified 62 members of the PP2 gene family in the B. napus genome with an uneven distribution across the 19 chromosomes. A phylogenetic analysis classified the BnPP2s into four clusters (I-IV), with cluster I containing the most members (28 genes) as a consequence of its frequent genome segmental duplication. A comparison of the gene structures and conserved motifs suggested that BnPP2 genes were well conserved in clusters II to IV, but were variable in cluster I. Interestingly, the motifs in different clusters displayed unique features, such as motif 6 specifically existing in cluster III and motif 1 being excluded from cluster IV. These results indicated the possible functional specification of BnPP2s. A transcriptome data analysis showed that the genes in clusters II to IV exhibited dynamic expression alternation in tissues and the stimulation of S. sclerotiorum, suggesting that they could participate in SD resistance. A GWAS analysis of a rapeseed population comprising 324 accessions identified four BnPP2 genes that were potentially responsible for SD resistance and a transgenic study that was conducted by transiently expressing BnPP2-6 in tobacco (Nicotiana tabacum) leaves validated their positive role in regulating SD resistance in terms of reduced lesion size after inoculation with S. sclerotiorum hyphal plugs. This study provides useful information on PP2 gene functions in B. napus and could aid elaborated functional studies on the PP2 gene family.

TIAM2 Contributes to Osimertinib Resistance, Cell Motility, and Tumor-Associated Macrophage M2-like Polarization in Lung Adenocarcinoma.

Background: Osimertinib-based therapy effectively improves the prognosis of lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor mutations. However, patients will have cancer progression after approximately one year due to the occurrence of drug resistance. Extensive evidence has revealed that lipid metabolism and tumor-associated macrophage (TAM) are associated with drug resistance, which deserves further exploration. Methods: An osimertinib resistance index (ORi) was built to investigate the link between lipid metabolism and osimertinib resistance. The ORi was constructed and validated using TCGA and GEO data, and the relationship between ORi and immune infiltration was discussed. Weighted gene co-expression network analysis based on the M2/M1 macrophage ratio determined the hub gene TIAM2 and the biological function of TIAM2 in LUAD was verified in vitro. Results: ORi based on nine lipid metabolism-related genes was successfully constructed, which could accurately reflect the resistance of LUAD patients to osimertinib, predict the prognosis, and correlate with M2-like TAM. Additionally, TIAM2 was found to increase osimertinib tolerance, enhance cell motility, and promote M2-like TAM polarization in LUAD. Conclusions: The lipid metabolism gene is strongly connected with osimertinib resistance. TIAM2 contributes to osimertinib resistance, enhances cell motility, and induces M2-like TAM polarization in LUAD.

[Phagocytosis of microglia in neurodegenerative diseases].

With the acceleration of the aging society, neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), have become a rapidly growing global health crisis. Recent studies have indicated that microglia-neuron interactions are critical for maintaining homeostasis of the central nervous system. Genome-Wide Association Studies and brain imaging studies have suggested that microglia are activated in early stage of neurodegenerative diseases. Microglia are specialized phagocytes in the brain. The discovery of a new phagocytic pathway, trogocytosis, suggests that there is a close interaction between microglia and surviving neurons. In this review, we summarize the important roles of microglia in neurodegenerative diseases, and further analyze the functions and molecular mechanisms of microglia phagocytosis and trogocytosis.

A global survey of the transcriptome of allopolyploid Brassica napus based on single-molecule long-read isoform sequencing and Illumina-based RNA sequencing data.

Brassica napus is a recent allopolyploid derived from the hybridization of Brassica rapa (Ar Ar ) and Brassica oleracea (Co Co ). Because of the high sequence similarity between the An and Cn subgenomes, it is difficult to provide an accurate landscape of the whole transcriptome of B. napus. To overcome this problem, we applied a single-molecule long-read isoform sequencing (Iso-Seq) technique that can produce long reads to explore the complex transcriptome of B. napus at the isoform level. From the Iso-Seq data, we obtained 147 698 non-redundant isoforms, capturing 37 403 annotated genes. A total of 18.1% (14 934/82 367) of the multi-exonic genes showed alternative splicing (AS). In addition, we identified 549 long non-coding RNAs, the majority of which displayed tissue-specific expression profiles, and detected 7742 annotated genes that possessed isoforms containing alternative polyadenylation sites. Moreover, 31 591 AS events located in open reading frames (ORFs) lead to potential protein isoforms by in-frame or frameshift changes in the ORF. Illumina RNA sequencing of five tissues that were pooled for Iso-Seq was also performed and showed that 69% of the AS events were tissue-specific. Our data provide abundant transcriptome resources for a transcript isoform catalog of B. napus, which will facilitate genome reannotation, strengthen our understanding of the B. napus transcriptome and be applied for further functional genomic research.

All-silica fiber-optic temperature-depth-salinity sensor based on cascaded EFPIs and FBG for deep sea exploration.

Using fusion splicing and hydroxide catalysis bonding (HCB) technology, an all-silica inline fiber-optic sensor with high-pressure survivability, high-resolution salinity measurement capability, and corrosion resistance for deep sea explorations is proposed and experimentally demonstrated. Two extrinsic Fabry-Perot interferometers (EFPIs) and a fiber Bragg grating (FBG) are cascaded in one single-mode fiber (SMF), enabling structural integration of single lead-in fiber and versatility of the sensing probe for temperature, depth, and salinity monitoring. The HCB technology offers a polymer adhesive-free assembly of one open-cavity EFPI for refractive index (RI) (salinity) sensing under normal pressure and temperature (NPT) conditions, showing obvious advantages of strong bonding strength, reliable effectiveness, and no corrosive chemicals requirements. The other EFPI formed by a fused structure is designed for pressure (depth) measurement. The cascading of EFPIs, especially the open-cavity EFPI immersed in water, will result in large light transmission loss and bring challenges to signal interrogation. Graded-index fiber (GIF) micro-collimators and reflective films are added to prevent dramatic degradations of signal intensity and fringe visibility underwater. Thereby, a Fabry-Perot (FP) cavity of several hundreds of microns in length and an open cavity of a thousand microns can be cascaded for underwater applications, effectively enhancing sensitivities and underwater signal readout simultaneously. Results show that the proposed sensor can well operate in the deep-sea pressure range of 0∼2039.43 mH2O, RI range of 1.33239∼1.36885 RIU, and temperature range of 23∼80 °C, with resolutions of 0.033 MPa, 4.16×10-7 RIU, and 0.54 °C, respectively. With the multi-parameter measurement capability, all-silica construction, and inline compact structure, the proposed sensor could be a potential candidate for deep sea exploration.

Rhombic optical path difference bias white light interferometric fiber-optic gyroscope.

We present a novel, to the best of our knowledge, white light interferometric fiber-optic gyroscope (IFOG) scheme using a fiber-optic rhombic optical path difference (OPD) bias structure to interrogate with a sensing coil to realize rotation rate measurement without a phase modulator. The OPD bias structure composed of four (2×1) 3 dB single-mode fiber couplers was constructed to implement non-reciprocal OPD bias. White light interferometric demodulation was utilized to acquire the change in OPD due to the Sagnac-phase shift. Absolute linear output can be obtained. We produced the principle prototype of rhombic OPD bias white light IFOG without utilizing a phase modulator. An experimental demonstration of the IFOG prototype system achieves linear output with respect to the OPD difference in detecting rotation rate.

dCA1-NAc shell glutamatergic projection mediates context-induced memory recall of morphine.

Opioid relapse is generally caused by the recurrence of context-induced memory reinstatement of reward. However, the internal mechanisms that facilitate and modify these processes remain unknown. One of the key regions of the reward is the nucleus accumbens (NAc) which receives glutamatergic projections from the dorsal hippocampus CA1 (dCA1). It is not yet known whether the dCA1 projection to the NAc shell regulates the context-induced memory recall of morphine. Here, we used a common model of addiction-related behavior conditioned place preference paradigm, combined with immunofluorescence, chemogenetics, optogenetics, and electrophysiology techniques to characterize the projection of the dCA1 to the NAc shell, in context-induced relapse memory to morphine. We found that glutamatergic neurons of the dCA1 and gamma aminobutyric acidergic (GABA) neurons of the NAc shell are the key brain areas and neurons involved in the context-induced reinstatement of morphine memory. The dCA1-NAc shell glutamatergic input pathway and the excitatory synaptic transmission of the dCA1-NAc shell were enhanced via the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) when mice were re-exposed to environmental cues previously associated with drug intake. Furthermore, chemogenetic and optogenetic inactivation of the dCA1-NAc shell pathway decreased the recurrence of long- and short-term morphine-paired context memory in mice. These results provided evidence that the dCA1-NAc shell glutamatergic projections mediated the context-induced memory recall of morphine.

The Protective Role of E-64d in Hippocampal Excitotoxic Neuronal Injury Induced by Glutamate in HT22 Hippocampal Neuronal Cells.

Epilepsy is the most common childhood neurologic disorder. Status epilepticus (SE), which refers to continuous epileptic seizures, occurs more frequently in children than in adults, and approximately 40-50% of all cases occur in children under 2 years of age. Conventional antiepileptic drugs currently used in clinical practice have a number of adverse side effects. Drug-resistant epilepsy (DRE) can progressively develop in children with persistent SE, necessitating the development of novel therapeutic drugs. During SE, the persistent activation of neurons leads to decreased glutamate clearance with corresponding glutamate accumulation in the synaptic extracellular space, increasing the chance of neuronal excitotoxicity. Our previous study demonstrated that after developmental seizures in rats, E-64d exerts a neuroprotective effect on the seizure-induced brain damage by modulating lipid metabolism enzymes, especially ApoE and ApoJ/clusterin. In this study, we investigated the impact and mechanisms of E-64d administration on neuronal excitotoxicity. To test our hypothesis that E-64d confers neuroprotective effects by regulating autophagy and mitochondrial pathway activity, we simulated neuronal excitotoxicity in vitro using an immortalized hippocampal neuron cell line (HT22). We found that E-64d improved cell viability while reducing oxidative stress and neuronal apoptosis. In addition, E-64d treatment regulated mitochondrial pathway activity and inhibited chaperone-mediated autophagy in HT22 cells. Our findings indicate that E-64d may alleviate glutamate-induced damage via regulation of mitochondrial fission and apoptosis, as well as inhibition of chaperone-mediated autophagy. Thus, E-64d may be a promising therapeutic treatment for hippocampal injury associated with SE.

Host Mesothelin Expression Increases Ovarian Cancer Metastasis in the Peritoneal Microenvironment.

Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLNKO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLNKO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLNWT) or MSLNKO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLNKO mice compared to MSLNWT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.

Miniature fiber-optic tip pressure sensor assembled by hydroxide catalysis bonding technology.

A miniature fiber-optic tip Fabry-Perot (FP) pressure sensor with excellent high-temperature survivability, assembled by hydroxide catalysis bonding (HCB) technology, is proposed and experimentally demonstrated. A standard single-mode fiber is fusion spliced to a fused silica hollow tube with an outer diameter (OD) of 125 µm, and a 1-µm-thick circular silicon diaphragm with a diameter slightly larger than the OD is bonded to the other endface of the hollow tube by HCB technology. The ultrathin silicon diaphragm is prepared on a silicon-on-insulator (SOI) wafer produced by microelectromechanical systems (MEMS), providing the capability of large-scale mass production. The HCB technology enables a polymer-free bonding between diaphragm and hollow tube on fiber tip with the obvious advantages of high alignment precision, normal pressure and temperature (NPT) operation, and reliable effectiveness. The static pressure and temperature response of the proposed sensor are discussed. Results show that the sensor has a measurable pressure range of 0∼100 kPa, which is well consistent with the measurement range of biological blood pressure. The pressure sensitivity is up to 2.13 nm/kPa with a resolution of 0.32% (0.32kPa). Besides, the sensor possesses a unique high-temperature resistant capability up to 600 °C, which can easily survive even in high-temperature sterilization processes, and it has a low temperature dependence of 0.09 kPa/°C due to the induced HCB bonding technology and the silicon-based diaphragm. Thus, the proposed fiber tip pressure sensor is desirable for invasive biomedical pressure diagnostics and pressure monitoring in related harsh environments.

Differential-pressure fiber-optic airflow sensor for wind tunnel testing.

A differential-pressure fiber-optic airflow (DPFA) sensor based on Fabry-Perot (FP) interferometry for wind tunnel testing is proposed and demonstrated. The DPFA sensor can be well coupled with a Pitot tube, similar to the operation of the differential diaphragm capsule in the airspeed indicator on the aircraft. For differential pressure sensing between total pressure and static pressure in the airflow, an FP cavity is formed between the sensing diaphragm and a fiber end-face, and a tubule is inserted into the FP cavity. According to the principle of differential pressure derived from Bernoulli's equation, the airflow velocity can be determined by monitoring the change of the FP cavity length. The experimental results demonstrate that a DPFA sensor with 0∼11 kPa measurable range, 826.975 nm/kPa sensitivity, and 0.008% (0.89 Pa) resolution can be realized. Combined with a 100 Hz-sweep frequency self-developed white light interferometric (WLI) interrogator and a Pitot tube, the DPFA sensor can be used for measuring the airflow velocity of 2.0∼119.24 m/s with an accuracy of 0.61%. The system is applied to the analysis of the flat-plate boundary layer, a wind tunnel experimental model, where the results are consistent with those of the theoretical analysis and from the standard electronic pressure transducer. With the large measurable range, high sweep frequency, and high precision, the system has potential application value for wind tunnel experimental investigation and in-flight measurement of airspeed.

Single-strand DNA-scaffolded copper nanoclusters for the determination of inorganic pyrophosphatase activity and screening of its inhibitor.

A fluorescence method for the determination of inorganic pyrophosphatase (PPase) activity has been established based on copper nanoclusters (CuNCs). The polythymine of 40 mer (T40) acts as a template for the reduction reaction from Cu2+ to Cu0 by ascorbic acid (AA). This reaction leads to the formation of fluorescent CuNCs with excitation/emission peaks at 340/640 nm. However, the higher binding affinity between inorganic pyrophosphate (PPi) and Cu2+ hinders the effective formation of CuNCs. This shows low fluorescence intensity. PPase catalyzes the hydrolysis of PPi into Pi during which free Cu2+ ions are produced. This facilitates the formation of fluorescent CuNCs. Thus, the fluorescence intensity was restored. The fluorescence enhancement of the system has a linear relationship with PPase activity in the range 0.3 to 20 mU·mL-1, and the detection limit is0.2 mU·mL-1. The relative intensity (I/I0) at 640 nm for the analytical solution versus system is also employed to screen the inhibitor for PPase with high efficiency. Graphical abstract Schematic representation of a fluorescent assay for the determination of inorganic pyrophosphatase activity and screening its inhibitor based on single-strand polythymine-scaffolded copper nanoclusters.

Large-Scale Image Analysis for Investigating Spatio-Temporal Changes in Nuclear DNA Damage Caused by Nitrogen Atmospheric Pressure Plasma Jets.

The effective clinical application of atmospheric pressure plasma jet (APPJ) treatments requires a well-founded methodology that can describe the interactions between the plasma jet and a treated sample and the temporal and spatial changes that result from the treatment. In this study, we developed a large-scale image analysis method to identify the cell-cycle stage and quantify damage to nuclear DNA in single cells. The method was then tested and used to examine spatio-temporal distributions of nuclear DNA damage in two cell lines from the same anatomic location, namely the oral cavity, after treatment with a nitrogen APPJ. One cell line was malignant, and the other, nonmalignant. The results showed that DNA damage in cancer cells was maximized at the plasma jet treatment region, where the APPJ directly contacted the sample, and declined radially outward. As incubation continued, DNA damage in cancer cells decreased slightly over the first 4 h before rapidly decreasing by approximately 60% at 8 h post-treatment. In nonmalignant cells, no damage was observed within 1 h after treatment, but damage was detected 2 h after treatment. Notably, the damage was 5-fold less than that detected in irradiated cancer cells. Moreover, examining damage with respect to the cell cycle showed that S phase cells were more susceptible to DNA damage than either G1 or G2 phase cells. The proposed methodology for large-scale image analysis is not limited to APPJ post-treatment applications and can be utilized to evaluate biological samples affected by any type of radiation, and, more so, the cell-cycle classification can be used on any cell type with any nuclear DNA staining.